Epitopes differential detection

The use of pairs of matched spectral variants of GFP, like cyan and yellow or GFP and mRed, to differentially tag proteins and look for interactions, is now in routine use. The approach can be readily applied to homodimerization of molecules that differ only by their living color or epitope tag. For example, homomultimer-ization of a viral coat protein can be observed by imaging a mixture of cyan and yellow tagged homomeric molecules [46], whereas oc-synuclein stacking can be detected by utilizing a-synuclein transcripts that encoded different epitope tags for detection by immunostaining [47],... 

It is relevant to explain the use of the letters CD as a prefix to monoclonal antibodies. Lymphocytes possess many different surface proteins, each of which possesses many distinct epitopes. To classify these lymphocyte antigens, a numbering system has been established that clusters molecules having similar epitopes. Thus, all monoclonal antibodies that detect the epitopes on a single antigen are assigned to a numbered cluster of differentiation (CD). In most cases a defined CD denotes a protein of specific function. For example, the protein called CD4 is associated with cells (lymphocytes) that help the immune response, while CD8 is found on cells that suppress the immune response. 

Some of the monoclonal antibodies mentioned below recognize different p53 molecule conformations. Also, detection of p53 with different antibodies depends on the time of its synthesis. It has been suggested that the p53 epitope for antibody 1620 remains cryptic immediately after synthesis in human keratinocytes and may not be exposed until late in the life of the protein (Spandau, 1994). Furthermore, different conformations of p53 may predominate in different differentiation stages of the cell or tissue. In addition, differentiation-specific cellular proteins and other proteins that may bind to p53 may mask epitopes on p53 at various stages of differentiation. For example, heat shock protein 70 is known to associate with p53 (Hainaut and Milner, 1992). 

To determine if differential glycosylation of fibronectin (Fn) in inflammatory synovial fluid (SF) included expression of an oncofetal epitope (Onf Fn) previously detected only on Fn derived from embryonal or neoplastic tissue [61], Fn was purified from plasma, SF and synoviocyte conditioned medium by affinity chromatography and analyzed by sodium dodeeyl sulfate-polyacrylamide gel electrophoresis and Western blotting using a monoclonal antibody (FDC-6) specific for the Onf Fn. The Onf Fn was not expressed on... 

Treatment with 25 //g/ml digitonin for 5 min does not permeabilize the peroxisomal membrane (differential permeabilization). Only epitopes on the cytosolic surface of the peroxisomal membrane can be detected. 

Thus, these initial proof-of-principle experiments demonstrate the ability to use the epitope specificity of antibodies to specifically detect a molecule of interest within the complex intracellular milieu of the cytoplasm, albeit with limitations. However, the small size of these probes did allow for membrane penetration within specific and distinct sub-cellular regions, which perhaps will permit differential localization of target molecules within a cell in the future. 

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